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Applied and https://doi.org/10.1007/s00253-018-9391-9

MINI-REVIEW

Bacteria as biological control agents of freshwater : is it feasible beyond the laboratory?

L. L. Ndlela1,2 & P. J. Oberholster1,2 & J. H. Van Wyk2 & P. H. Cheng1

Received: 27 July 2018 /Revised: 7 September 2018 /Accepted: 9 September 2018 # Springer-Verlag GmbH Germany, part of Springer 2018

Abstract Biological control of cyanobacteria is a -researched area with a central focus on laboratory-scale studies. Numerous reports have been made on algicidal isolates, with as a major component of the antagonists. The research in this review draws a brief summary of what is currently known in the area of freshwater cyanobacteria being inhibited by bacterial isolates. , and are among the most commonly reported phyla of bacteria associated with or employed in this research area. However, there are limited reports of upscaling these control measures beyond the laboratory scale. Lytic control agents are the most commonly reported in the literature with subsequent release. From a perspective, this is not feasible. Based on the available literature, temperature, pH and nutrient changes have been explored in this short review as possible contributors to less optimal bacterial performance. Moreover, the investigation into optimising some of these parameters may lead to increased bacterial performance and, therefore, viability for upscaling this biological control. Through the compilation of current research, this review offers insight to live predator-prey interactions between cyanobacteria and algicidal bacteria.

Keywords Algicidal bacteria . Cyanobacterial inhibition . Biological control . Harmful algal blooms

Introduction they pose a threat to human and health, but they also present a challenge in terms of facilities The current research will assess the feasibility of bacteria as (Ndlela et al. 2016). A review by Paerl et al. (2016) has indi- cyanobacterial control agents beyond the laboratory. This will cated the significance in developing mitigation measures for be based on consolidation of the existing information on bio- cyanobacterial blooms, especially since they will be more dif- logical control of cyanobacteria, based on the reviews of Sigee ficult to curb due to increased temperatures caused by et al. (1999) and Gumbo et al. (2008)aswellasnumerous change in the future (Paerl et al. 2016). other publications on this topic. The focus will be more spe- The concept of biological control is the use of natural en- cifically in the area of bacteria as a biological control agent to emies to control a target , and among the measures living cyanobacterial cells in freshwater environments (Sigee previously mentioned in a review by Sigee et al. (1999), is the et al. 1999; Gumbo et al. 2008). use of bacteria as a means to control cyanobacterial cells. In With the rise in cyanobacterial bloom occurrences and tox- the broader focus of biological control, a review by icity, their mitigation is a crucial area of research. Lakes from Verschuere et al. (2000) thoroughly investigated the use of across the continents in the first world and developing coun- bacteria as possible biological control agents. The tries alike have been plagued with these blooms. Not only do key modes of action listed by bacteria as methods of biolog- ical control indicate antagonism as the more common mode of action in (Verschuere et al. 2000). * L. L. Ndlela In the study of bacteria as control agents in aquaculture, a [email protected] variety of inhibitory compounds such as , bacterio- cins, and lysosomes is produced by probiotic 1 Council for Scientific and Industrial Research, 11 Jan Celliers Road, bacteria. Whilst controlling other , these com- Stellenbosch 7600, South Africa pounds can have a positive growth effect as well on unicellular 2 Department of and , Faculty of Science, Stellenbosch algae and this needs to be clearly understood prior to University, Matieland 7600, South Africa Appl Microbiol Biotechnol implementation of certain as biological control agents Control agents associated with cyanobacteria (Verschuere et al. 2000). and the susceptibility of the target Previous studies employing the use of bacteria in curbing cyanobacteria have been conducted primarily at laboratory scale, with focus on a dominant species within a bloom or An extensive review (Van Wichelen et al. 2016) describes the on cultures of a given cyanobacterial isolate susceptibility of sp. to a variety of control agent (Nakamura et al. 2003; Choi et al. 2005; Jung et al. 2008; ranging from to fungi and bacteria. The lack of appli- Shao et al. 2014;Suetal.2016a). These studies seldom ac- cation of this form of biological control outside laboratory count for the mixed cultures of or the possible conditions is also mentioned. This is of concern as a number variations in temperature during the exposures under lab con- of possible control measures with living organisms have been ditions or the fluctuations within the . A explored with minimal upscaling opportunities, with recom- recent study has found some of the interventions in curbing mendations made against upscaling beyond the lab in some cyanobacterial blooms were not as effective as reported, with studies (Kim et al. 2008). a strong caution to critically evaluate these control measures The first challenge comes from the lack of information on among which bacteria, artificial mixing and algae were men- the type of bacteria and interactions among species that may tioned (Lürling et al. 2016). hinder how effective a given control agent may be under Recent research by Demeke (2016) describes the use of conditions as opposed to laboratory-based findings. The di- metabolites from the bacteria Flexibacterium in the control versity and intercellular interactions are numerous to be ade- of the filamentous cyanobacteria and the lytic quately accounted for. To quantify or individually explore activity of the bacteria against the cyanobacteria these factors is complex and requires further intensive re- flos-aquae (Demeke 2016). These are just a search to a more conclusive picture. Therefore, although few of many examples and show the interest and potential in there are numerous studies and reports of effective microor- the area of biological control. Over the past decade, advance- ganisms applicable in laboratory studies (Choi et al. 2005; ments in the use of bacteria as cyanobacterial control agents Jung et al. 2008; Kim et al. 2008; Zhang et al. 2016), none are not as extensive. of these have been conclusively upscaled or pursued beyond the lab due to the myriad of uncertainties that present them- selves within a mixed population. Another question that arises Aim of research is how well do these control agents regulate their algicidal characteristics in the natural environment within a mixed mi- crobial population? Taking a closer look at Microcystis spe- Although not exhaustive, this research aims to establish a cifically which has the most available literature and is the timeline of developments in this mode of cyanobacterial con- causative agent of the most common toxic blooms, if trol and what is currently known, with the intention of collat- cyanobacteria, particularly Microcystis, present such a wide ing pitfalls and possible future research challenges. The study susceptibility to environmental isolates, how they are able to is focused on the viability of this biological control and which continue proliferating and creating toxic aquatic conditions? factors could be optimised for further progress, based on the Table 1 presents a synopsis of the known types of control available literature. agents and the type of cyanobacterium they have a predatory or lytic impact on, exclusive of bacterial isolates from various studies (Zhang et al. 2008;Wangetal.2011; Gao et al. 2012a; Data collection approach Mialet et al. 2013; Mohamed et al. 2014; Leitão et al. 2018). The use of bacteria as control agents will be discussed in more The literature used within this mini-review was collected from depth in this review. the following databases: Google Scholar, Scopus and Another area of interest is the form of species, such as EBSCOhost with the following key words: Balgicidal Microcystis, as laboratory strains and their natural form. bacteria^, BMicrocystis/Oscillatoria// Generally, under natural conditions, these cells present as /Aphanizomenon inhibiting bacteria^, Bbiological large colonial isolates with layers, surrounded by control of cyanobacteria^, Balgicidal bacteria^ and Bbacteria various microorganisms, which have been believed to have lysing cyanobacteria^, with the frame delineated to symbiotic relations with the cyanobacteria. This colonial form 2000–2017. Further delineation was done based on focusing is not often observed in laboratory strains that present as uni- on freshwater-related studies and the /suppression/control cellular, and a recent study (Geng et al. 2013) found that of living cyanobacterial cells by living bacterial agents, with laboratory-grown strains are not able to revert back to colonial minimal focus on microcystin degraders and bacterial metab- form after subculturing within the laboratory and they tend to olites employed to control whole cyanobacterial cells. form two-celled or unicellular cells, which offer reduced Appl Microbiol Biotechnol

Table 1 Summary of the reported control agents against cyanobacteria

Control agent Target organism Mode of action Reference

Cyanophage sp. and Lytic dependent on populations Wang et al. (2011); Gao et al. agardhii Cell lysis (2012a) Golden algae Microcystis sp. Direct grazing and microcystin Zhang et al. (2008) Poterioochromonas degradation Bdelloid Cyanobacteria and Grazing Mialet et al. (2013) Notodiaptomus iheringi Microcystis sp. Grazing Leitão et al. (2018) Trichoderma citrinoviride Microcystis sp. Indirect lysis and microcystin Mohamed et al. (2014) degradation

resilience as opposed to colonies. This differential response is bacterial communities found that the phyla Proteobacteria also observed in lab studies when exposing both forms of and Bacteroidetes were among the most common phyla in cells. Therefore, one may consider the study of biological blooms of cyanobacteria Microcystis and Anabaena sp. control of unicellular cells to not be fully indicative of the These phyla were also found in various blooms in Swedish possible response in a natural . An earlier study by lakes (Eiler and Bertilsson 2004). Various studies have been Yang et al. (2006) found that colonial Microcystis were more conducted in understanding cyanobacterial blooms and, there- successful at warding off grazing by cladocerans as opposed fore, the bacterial communities associated with them. to the unicellular isolates, which indicates a possible mecha- Naturally, cyanobacteria exist as co-cultures with a variety nisms of defence that is not often noted with tested unicellular of . When assessed, Shi et al. (2011) and Cai isolates employed in the laboratory. In the findings of a recent et al. (2014) found that there are aggregates of free-living study, these cells can morph into various morphotypes of bacteria as well as attached bacterial species which are within Microcystis within colonies, due to the changing environmen- the mucilage of colonial Microcystis cells. The bacterial diver- tal conditions within the waterbody. This indicates a steady sity has been found to change with the different stages of a shift of Microcystis species, and these are not often observed bloom (Shi et al. 2011) and with various species (Bagatini in targeted biological control efforts under controlled condi- et al. 2014). Some phyla are in higher abundance at the deg- tions. The study further indicates the value of this defence radation phase of a bloom, whilst some are more represented mechanism to a given species, hence the value in a control at the start or peak of blooms. The fact that these microorgan- agent that is effective within a mixed population and also has isms freely exist within bloom conditions makes them ideal enough host specificity to remain effective against the targeted control agents, although it is understood that certain classes cluster of organisms (Man et al. 2018). Moreover, more evi- aid the proliferation of cyanobacteria (Tian et al. 2007;Shi dence of the natural prevalence of the cells as colonies has et al. 2009). been supplied in a more recent literature review on the colony From the above-mentioned literature studies, formation characteristics of Microcystis (Man et al. 2018). , and Bacteroidetes Table 1 summarises the commonly reported antagonists are the more closely associated and reported species associat- from different trophic levels against cyanobacteria and how ed with non-axenic cyanobacterial aggregates. This applies to these predators commonly control cyanobacterial isolates. studies conducted on Anabaena, Aphanizomenon, Cylindrospermopsis and Microcystis cultures. A study by Cai et al. (2014) found similar phyla associated with Heterotrophic bacteria associated Microcystis,withAlphaproteobacteria most abundant in the with cyanobacterial blooms small cell aggregates and Firmicutes most abundant in the larger cyanobacterial cell aggregates. Louati et al. (2015)re- Apart from the known characteristics of most bacteria and ported that bacterial communities differed based on the their robust growth, being non-fastidious and ubiquity, they cyanobacterial blooming at a given time and that there is a form an integral part of the diversity in algal blooms (Eiler and beneficial relationship between cyanobacterial attached bacte- Bertilsson 2004). This is significant in assessing whether the ria, seen in the accelerated growth of non-axenic commonly reported control agents fall into the phyla most cyanobacteria as compared to axenic strains. Firmicutes was commonly indicated in cyanobacterial control. More impor- also reported to be in lower abundance in this study as com- tantly, this may establish whether there is any link between pared to Proteobacteria and . With Bacillus sp. these specific control agents and how closely they are ob- isolates being commonly reported as algicidal bacteria, it is served in the cyanobacterial . An assessment of interesting to note that they are not abundant in the smaller Appl Microbiol Biotechnol cyanobacterial cell aggregates, which are in closer proximity properties at higher temperature, supporting the findings that to the cyanobacteria. most isolated clinical bacteria are mesophilic in nature (Hajdu Free-living and cyanobacterial attached bacteria have been et al. 2010). Although a variety of factors influence the growth differentiated based on the aggregates they form after filtration rate of bacteria in conjunction with temperature, a similar and separation from each other. The cells found closest to the phenomenon of temperature influencing growth rates in a cyanobacterial cells’ mucilage are thought to have a mutual- number of studies (Pietikäinen et al. 2005). istic beneficial relation or considerable tolerance to the given Rampelotto (2013) considered cyanobacteria as being the species (Bagatini et al. 2014). This is supported by an earlier most resilient to temperature changes among the , study conducted by Bouvy et al. (2001), where the with their discovery and viability occurring in a wide expanse cyanobacteria Cylindrospermopsis raciborskii did not impose of environmental conditions. Almost two decades ago, re- a major change in or specific species suppression search on the effect of lowered temperatures on in the bacterial or zooplankton community within a and psychrophilic bacteria indicated that the affinity for se- study in Brazil. The indication of the bacterial diversity in questering or binding given substrates was lowered at de- cyanobacterial aggregates in nature is that the more beneficial creased temperatures. There was a noteworthy difference in bacteria are found in closer proximity to the cyanobacteria. the cell affinity (Nedwell 1999), with respiratory rate inhibi- This may explain the difficulty in natural bloom control, as tion even in , when temperatures were lowered predatory isolates would be lower in abundance, with a re- beyond the optimum (Pomeroy et al. 1991). Temperature duced contact time to the targeted cyanobacterium. changes of 10 °C showed a distinct decrease in cell affinity for mesophiles (Nedwell 1999). Movahedi and Waites (2002) further showed that cold resulted in a expression Factors associated with cyanobacterial change in sporulating Bacillus cells, compared to an earlier control that may be optimised study showing a growth lag in vegetative cells after tempera- ture drops of about 20 °C (Graumann and Marahiel 1999). If bacteria are to be applied on a larger scale as control agents, From this research, although resilience may be increased in some factors would require optimisation. From previous liter- further generations, it is clear that drastic temperature changes ature, this review has identified temperature, nutrients and pH are not ideal in the optimal performance of a mesophilic mi- as parameters that could be amended for improved bacterial croorganism. To provide a bit of contrast, a study on the heat performance that may be viable on a larger scale, beyond the shock response of E. coli cells showed that a 12 °C tempera- laboratory. These parameters were easily identified as they ture increase to 42 °C subsequently increased the growth rate were the most often recorded in the studies included here. of these cells, whilst higher temperatures of 48 °C to 55 °C Although these are not the only key factors to be considered, indicated an initial increase and, thereafter, inhi- they are the most frequently documented. bition (Soini et al. 2005). However, it appears that temperature changes beyond 10 °C were induced in most experimental cases where bacteria are isolated to control cyanobacteria. Temperature differences and their possible Although the studies here include clinical isolates, the trend implications of lowered growth rate at lower temperatures for mesophiles is clear. Numerous studies on algicidal bacteria have been conducted Viewing this point in the context of African waters, Ndlela under laboratory conditions. The algicidal bacteria are often et al. (2016) found water temperatures during blooms which cultured under different nutritional and temperature condi- can range from above 30 °C to as low as 10 °C. A South tions, slightly higher than those of the targeted cyanobacteria. African study by Gumbo et al. (2010) on the biological control This might be one of the factors to consider for increased of Microcystis reported the of algicidal Bacillus bacterial efficiency in controlling blooms on a larger scale. mycoides using the plaque assay, with subsequent exposure There is considerable evidence that growth temperatures sig- using this Bacillus as a control organism. Cultures were grown nificantly impact the protein activity (Patke and Dey 1998)in separately, with bacterial isolates at 37 °C, whilst the target microorganisms, with a proven increase in growth rate at tem- cyanobacteria were kept at ambient temperature in a different peratures beyond 30 °C for Bacillus, coli, medium; thereafter, the control agent was co-cultured with the and sp. strains (Membré et al. 2005). cyanobacteria at a 1:1 volume ratio and observed over 6 days Studies on the effects of temperature on bacterial isolates have (Gumbo et al. 2010). been modelled and better understood from as far back as the Research from Oberholster and Botha (2010) analysing the 1980s (Ratkowsky et al. 1982; White et al. 1991). A 2010 hyper scum and of Microcystis sp. in Lake report assessing clinical isolates of sp. also Hartbeespoort showed that over a period of 6 months at the found increased populations and peak of the bloom, temperatures ranged between 23 and Appl Microbiol Biotechnol

25 °C. This is further supported by the findings of the research This is seen in many of the lab-scale studies listed in this in Theewaterskloof, where a bloom occurred at temperatures article, with the general approach being to enhance the pred- as low as 14 °C (Oberholster et al. 2015). Another study by ator through rich media and higher temperatures. This begs Oberholster et al. (2009), assessing animal mortalities to the question whether the conditions employed would result in microcystins, also indicated that the average water tempera- effective biological control in the natural environment. ture between 2005 to 2006 was 25.1 °C in Lake Furthermore, how may one feasibly upscale this technology Hartbeespoort, over that period, with a clear indication that in an aquatic system without major hampering of the biodi- temperature increases of around 0.84 °C over the years due versity? Another question that arises is whether the response to climate change have influenced the toxicity and prevalence to climate changes and therefore increased nuisance algal of cyanobacterial blooms (Oberholster et al. 2009). blooms is eradication, suppression or removal. In light of this information among other reports, why is the Although there are numerous papers reporting the use of temperature of control agents seldom uniform to blooms con- microcystin-degrading bacteria, exclusive of live cell-to-cell sidered in the lab-scale implementation of biological control? interaction, which indicate the metabolites and that Listed in Table 2 are the different temperatures that bacteria lead to cyanobacterial and inhibition (Ji et al. 2009;Luo and target cyanobacteria are grown under, based on previous et al. 2013;Yangetal.2014), the focus of this review is on live research (Manage et al. 2000;Choietal.2005;Muetal.2007; cell interaction between the predator and the targeted Ren et al. 2010;Yangetal.2012; Lin et al. 2014; Zhu et al. cyanobacteria. 2014;Suetal.2016a, b;Zhouetal.2016). Only research over the last few years indicates a consideration of temperature similarity and, in some cases, nutrient acclimatisation as in Alkalinity and acidity: the other factors the case of Shao et al. (2014) in lab-scale studies. Marked in in biological control red are temperature differences that indicate a difference great- er than 10 °C which may induce the previously mentioned Numerous studies indicate a preference for cyanobacterial cold shock in bacterial isolates. proliferation at more alkaline conditions (Gumbo et al.

Table 2 Comparison of temperature and media Isolate Media Temp (°C) Reference differences between bacteria and target cyanobacteria Bacteria denitrificans Casitone 34 Manage et al. (2000) neyagawaensis Nutrient broth 40 Choi et al. (2005) Bacillus fusiformis Nutrient broth 30 Mu et al. (2007) aeruginosa R219 Luria-Bertani 37 Ren et al. (2010) sp. Nutrient broth 23 Yang et al. (2012) Bacillus sp. Modified CT medium 28 Shao et al. (2014) sp. extract peptone 30 Lin et al. (2014) Nutrient broth 30 Zhou et al. (2016) sp. Luria-Bertani 30 Su et al. (2016a) sp. Luria-Bertani 30 Su et al. (2016b) Cyanobacteria Microcystis spp. MA medium 25 Manage et al. (2000) CB medium 25 Choi et al. (2005) Various cyanobacteria BG-11, etc. 16–24 Mu et al. (2007) Microcystis aeruginosa BG-11 28 Ren et al. (2010) Microcystis aeruginosa MA medium 23 Yang et al. (2012) Microcystis aeruginosa CT medium 28 Shao et al. (2014) Microcystis aeruginosa BG-11 25 Lin et al. (2014) Microcystis aeruginosa BG-11 25 Zhou et al. (2016) Microcystis aeruginosa BG-11 25 Su et al. (2016a) Microcystis aeruginosa BG-11 25 Su et al. (2016b)

Values in italics are temperature differences that indicate a difference around 10 °C Appl Microbiol Biotechnol

2008), with a pH of as high as 10 being optimal for the pro- competition for phosphorous, as indicated in a study by liferation of blooms (Gao et al. 2012b). A study on the mass Vadstein et al. (2003) in a microcosm, was the limiting nutri- culturing of for its multiple benefits in industrial ent in the co-existence of algae and heterotrophic bacteria in applications found that higher pH conditions were primarily an aquatic system, whilst the availability of organic useful in warding off potential predators, as these maintain an was greatly contributed by algal predators (rotifers) than the advantage to grow at more alkaline conditions in comparison algae themselves. Moreover, if we consider the research find- to other freshwater species that graze on it. Optimal growth ings of Ji et al. (2017) when assessing nutrient competition did not differ much from neutral pH up to a pH of 10.5, which between eukaryotic algae and the cyanobacterium greatly limited the growth capacity of the grazer. Given that Microcystis, we find that at the typical high pH and low CO2 alkalinity is higher under natural bloom conditions and that conditions, eukaryotic algae are capable of competing at these over continuous culture, there is often an increased pH level conditions. Since the depletion of by created by the presence of cyanobacterial cyanobacteria under bloom conditions has been a hampering (Touloupakis et al. 2016), and it stands to a reason that control impact to the photosynthetic activities of other phytoplankton, agents need to be alkalotolerant or maintain circum-neutral pH this is an interesting finding. However, the nutritional require- conditions when exposed to the target cyanobacteria. Another ments of the differ in the need of organic carbon, + earlier study found optimal growth of filamentous Schizothrix noted also in their uptake of NH4 from the nitrogen fixer calcicola at pH levels between 6 and 8. More acidic condi- Aphanizomenon (Adam et al. 2016), which makes use of N2 tions appeared to limit the pigment expression of this cyano- under limited nitrogen conditions. Heisler et al. bacterium (West and Louda 2011). Based on the literature (2008) also describe the ability of some species of referred to earlier in this study on the varied temperatures of cyanobacteria to shift to organic sources of nutrients, which the control agent and prey, the culture media referred to in means that in some blooms, this is a direct competition for most studies are neutral, with these conditions favourable for nutrients between the cyanobacteria and the bacteria. Perhaps most mesophiles. Therefore, although the value of alkalinity is under bloom conditions, the limited nutrients to all microor- crucial under proliferation, there is limited infor- ganisms shift the towards as a means to mation on the changes brought about by the control agent survive. The (exopolysaccharide) EPS layer of Microcystis from this perspective under laboratory test conditions and the cells of filamentous cyanobacteria are viable nutrient reviewed for the purposes of this report. Do heterotrophic sources for heterotrophs with a limited source. Therefore, bacteria lower the pH in the cyanobacterial culture medium? it may be that the bacterial predation of cyanobacteria is af- Is this also one of the changes that occurs in the growth me- fected by numerous factors including the limitation of nutri- dium under lab conditions to limit cyanobacterial growth? ents under bloom conditions.

Nutrient competition: some thoughts Previously reported bacterial control agents and ideas Among the literature, a number of bacterial control agents Nutrient competition under algal bloom conditions is vital in have been discovered. Figure 1 summarises a brief timeline enabling the domination of the cyanobacterial cells. The gas of bacteria reported to control cyanobacteria and/or algae. vesicle of isolates of Microcystis, for example, enables regu- Although the list is not exhaustive, it is clear that there are lation through the water column as well as light shading of common species reported against the cyanobacteria other phytoplankton through the formation of surface scum Microcystis specifically. In correlation with the findings on colony aggregates. As Paerl et al. (2016) indicate, the bacterial communities during a bloom, Pseudomonas, survival and competition strategies of this group of , and Alcaligenes as shown in prokaryotes are quite impressive. Therefore, the issue of Fig. 1 are isolates under the Proteobacteria , which nutrient competition in biological control using heterotrophs is abundant in cyanobacterial bloom communities, as previ- is a crucial consideration. Being mostly heterotrophic, the ously discussed. Streptomyces (Phankhajon et al. 2016)is response to reduced nutrients in the presence of within the phylum Actinobacteria, which has also been report- cyanobacterial dominance may be a potential trigger to the ed among the abundant phyla. Of interest, however, are the bacterial predator response to the cyanobacterial cell. This reports on Bacillus, which is within the Firmicutes phylum. may mean that at low nutrients, which are under the peak of This has been less frequently reported to be in abundance as bloom conditions or when the bloom is dominating the opposed to the other phyla during a cyanobacterial bloom, diversity in the aquatic system, the nutrient limitation gives although Shi et al. (2009) have mentioned this phylum among way to microcystins or the cyanobacterial cell being a those within a bloom. Moreover, in the study by Cai et al. plausible nutrient source for heterotrophic bacteria. The (2014), Firmicutes was found in the larger cyanobacterial cell Appl Microbiol Biotechnol

Fig. 1 Research timeline of findings related to algicidal bacteria from the or inhibited. Most of the current research is against Microcystis sp., with year 2000. Findings from each author are separated by a semicolon, with fewer publications against other species a summary of the bacterial and the cyanobacteria found to be lysed aggregates. The algicidal effect of Aquimarina isolates in a in causing lysis or inhibition of specific cyanobacteria based previous study also confirms the presence of Bacteroidetes on the available literature. phyla within the water column where the cyanobacterial From Table 3, it is evident that most of the research has bloom occurred (Chen et al. 2011). Although these species been conducted at laboratory scale (Fraleigh and Burnham exist within the bloom diversities, other studies have implied 1988;Shunyuetal.2006;Chenetal.2011; Zhang et al. that the species attached to the cyanobacterial cells have a 2011;Tianetal.2012; Luo et al. 2013; Jia et al. 2014). The symbiotic relationship with each other (Shi et al. 2011). In effective numbers range from as little as a million bacterial the same study, and sp. cells up to a 100 million, with effective ratios as low as 1:100 were also reported to be positively associated with all the way up to 100:1 of the control agent. The laboratory- Microcystis, serving as a potential protection, whilst the other scale studies show that the Proteobacteria phylum is one of organisms were detected during the vigorous period of the the more commonly reported bacterial control agents, and this Microcystis bloom. supports the findings of Manage et al. (2009), who first re- Mayali and Azam (2004) reviewed marine algicidal bacte- ported on external microcystin degraders which were outside ria, which interestingly indicated similar phyla such as those this phylum, isolated within the UK. The suppression was discussed in freshwater studies mentioned here. Repeatedly, based on cell lysis or growth limitation higher than 80% of earlier studies found that contact time is a significant factor in the starting populations (Manage et al. 2009). the effective control or lysis of cyanobacteria by predatory The earlier findings of Proteobacteria as the most common bacteria. Understanding why these control measures are not control agents may also be supported by the microbial diver- always effective on their own to curb blooms leads to a co- sity and abundance during a bloom, as it has been established nundrum of factors. Among the recent literatures, it appears that, at the peak of the bloom, Proteobacteria and that the phylum Firmicutes has several effective predatory Bacteroidetes are abundant. Algicidal microcystin degraders, isolates, especially from the Bacillus . This is interesting however, were also commonly reported from Firmicutes,par- to note as this phylum is generally found in lower abundance ticularly from Bacillus (Mayali and Azam 2004; Shunyu et al. under bloom conditions. 2006; Pei et al. 2007; Zhang et al. 2011), which may be logical In addition, most of the biological control implementations if Firmicutes increases towards the decline of a bloom. have been under small-scale laboratory conditions (Choi et al. In the case of liquid cultures, which is the most commonly 2005), with most culture conditions favouring the prolifera- reported experimental procedure, the predator bacterium is tion of the predator, usually at mesophilic conditions (30 °C), either isolated from within the bloom waters or seldom exter- whilst the cyanobacteria are cultured at ambient temperatures. nally sourced. Screening of primary inhibition is conducted, This is often under different nutrient and temperature condi- and based on this, further research is done to analyse algicidal tions, which are not always representative of cyanobacterial activity and the effects. The predator and prey are grown un- bloom conditions in nature. der separate conditions, with the cyanobacteria grown as axe- Table 3 summarises a few examples of the control - nic or non-axenic strains at ambient temperature, on BG-11, ism used as well as the effective ratios of the predator to prey Z8 or similar culture media (Nakamura et al. 2003). The Appl Microbiol Biotechnol

Table 3 Effective ratios required of predator bacteria to prey cyanobacteria in previous studies

Phylum/class Control organisms Target cyanobacteria Effective ratios (b/a)* Scale Source (ml)

Actinobacteria Streptomyces Microcystis aeruginosa 1 ml:99 ml 100 Luo et al. (2013) Proteobacteria/Alphaproteobacteria Xanthobacter Microcystis aeruginosa 1×108:2.2 × 106 50 Kim et al. (2008) autotrophicus Bacteroidetes Aquimarina Microcystis aeruginosa 4 ml:36 ml 150 Chen et al. (2011) Proteobacteria/ and Phormidium 5×106:1 × 108 100 Fraleigh and Burnham (1988) Firmicutes Bacillus fusiformis Microcystis aeruginosa, 3.6 × 107:412 μg/l 1000 Mu et al. (2007) Chlorella, chlorophyll Scenedesmus Oscillatoria 8 ml:80 ml dry weight 100 Jia et al. (2014) Microcystis aeruginosa 1.5 × 106:1 Tian et al. (2012) Aphanizomenon 5ml(1×108 cells):45 ml 50 Shunyu et al. flos-aquae (2006) Proteobacteria/ Pseudomonas aeruginosa Microcystis aeruginosa 40 μg/ml extract:2 × 107 1Renetal.(2010) Microcystis aeruginosa 1×108:1.2 × 107 150 Zhang et al. (2011) Microcystis aeruginosa 20 ml:20 ml 40 Gumbo et al. (2010)

*Bacteria:algae/cyanobacteria predator is most often grown at higher temperatures, in a dif- abundance or death. Flow cytometry has been recommended ferent medium. Cells of the predator are then introduced to the by Gumbo et al. (2014) as well as Cellamare et al. (2010). cyanobacterial population, and cell death or lysis is measured Most recent is the study conducted by Chapman (2016), microscopically or using similar methods as listed above. This where flow cytometry is employed as a predictive measure shortfall usually is not representative of the natural conditions for blooms, among the known technologies. within the environment as most cultures are axenic The typical experimental set-up involves the culturing of cyanobacteria. Table 3 shows the different experiments that mostly axenic or non-axenic cyanobacterial cultures, which have been applied in the control of freshwater cyanobacteria. are grown to a certain point, and thereafter, the predatory isolate is added at the optimised working ratios to achieve cell suppression. Most of these effective agents are of the Different conditions of biological control Proteobacteria or Firmicutes genus, often tested against implementation using bacteria Microcystis aeruginosa. The findings indicate cell lysis, which is often associated to increased toxicity of the test wa- Using Fig. 1 as a timeline guide of recent studies conducted in ters. As a result, most of the studies are not suitable for this area, there are a few general conditions that are applied in upscaling purposes or further development as is. The other previous studies to study algicidal or inhibitory effects of bac- findings indicate predator-to-prey ratios of at least 1:1, for terial isolates on targeted cyanobacteria. On a laboratory scale, reasonable cyanobacterial suppression and/or lysis. the studies are conducted primarily through Research by Su et al. (2016a, b)asreferredtoinTable2 indicated overall toxin suppression, which is not commonly & Plaque assays on solid medium (Rashidan and reported, although this is against an axenic culture of 2001; Gumbo et al. 2010) Microcystis. & Liquid medium growth assays (Nakamura et al. 2003; Based on the available literature, adaptation of this particular Choi et al. 2005; Gumbo et al. 2010) control method (i.e. bacterial biological control of cyanobacteria) & Flow cytometric assessments of cell viability and death is approached with caution, simply due to the numerously report- through live and dead stains, and cell surface ed effects of lysis as well as the unknown further biological monitoring (Cellamare et al. 2010; Zhou et al. 2012; impacts. In-depth reviews of the contact time and lysis mecha- Gumbo et al. 2014) nisms have indicated that no prescribed method may be effective against varied biodiversity (Gumbo et al. 2008). A more recent The methods listed above are among the most commonly synopsis of biological control in marine environments equally used, with chlorophyll measurements being the primary mea- stresses the point of uncertainty in the possible outcomes of surement of cell death. With Microcystis specifically, this has implementation as opposed to terrestrial biological control exper- proven to be difficult as it is not an accurate estimation of cell iments. Moreover, augmentative biological control appeared to Appl Microbiol Biotechnol be a more feasible projection compared to the introduction of broken down by the mlr , of which mlrA is responsible non-indigenous species (Atalah et al. 2015). for the primary breakdown of microcystins. The review Avery interesting 2017 report (Osman et al. 2017)assessed continues to indicate the suitability of microbial aggregates in the interactions of axenic cyanobacterial cultures in the pres- filter systems for microcystin degradation. Interestingly, the ence of known cyanolytic heterotrophs. The analysis of author also points towards the use of probiotic bacteria such as upregulation and downregulation indicated stress from both species for microcystin degradation. The use of the heterotrophs and the cyanobacteria from a nutrient com- other classes of probiotic bacteria in the EM mud balls as petition perspective as well as from a growth limitation aspect previously referred to in the report by Lürling et al. (2016)was, for Microcystis sp.However,theAphanizomenon flos-aquae however, not as successful in controlling Microcystis, at a living isolate tested under similar conditions indicated cell damage cell-to-cell interaction approach. It is also interesting to note, over a 96-h period but there were no clear indications of however, that microcystins in the environment are degraded growth limitation. The study led to the findings that the cell within 5–21 days, although most lab-scale studies have periods stress response was elicited by the attachment and aggregation beyond the minimum 5 days set to observe cyanobacteria or of the predatory heterotroph to the cyanobacteria as well as toxin suppression (Welker et al. 2001). extracellular compounds such as antibiotics, which affect pho- A compilation of some mitigation and prevention strategies tosynthesis in the cyanobacterial cells. Of further interest at field-scale level has indicated the control of nitrogen and among the many findings in this research is the expression phosphorus ratios in shallow and deep lakes among the pos- of , a protection from toxic peroxides and up- sible measures that can be implemented after analysis of the regulation of cold shock proteins by some of the bacterial lake system. From a biological intervention perspective, food isolates. The study also found an web management through mussels, which improve light con- shift in the bacterial isolates, indicating nutrient-related stress. ditions for other macrophytes, and clearing waters through These findings corroborate the theories in this review and are a grazing on cyanobacteria are described as a potential interven- key contribution to understanding the complex cell-to-cell in- tion. Moreover, the authors state earlier in their report that the teractions between the cyanobacterial isolates and their het- management has minimal impacts on bloom control erotrophic bacterial prey. This study was conducted on labo- (Stroom and Kardinaal 2016). Interestingly, the use of bacteria ratory axenic strains of cyanobacteria. is not included in these interventions and it may be due to the limited reports on conventional methods for controlling nutri- ent loading and bloom occurrence in field studies. This really Are there stories of success? draws attention towards further development of effective predatory bacterial application in bloom mitigation or preven- Thus far, an assessment of cyanobacterial control methods has tion, which is a key point of this review. found an application of effective microorganisms (EMs), Another publication by Park et al. (2017) indicates how which comprises of a mud ball of microbial consortia to be most of the studies in controlling cyanobacterial blooms have ineffective in population reduction of lab and wild strains of been under controlled laboratory conditions. However, the Microcystis, with effective suppression achieved at very high authors propose ultra-sonication, another abiotic measure as numbers, possibly due to other factors. It is interesting to note a method for bloom control, with compiled reductions of up to that , and Saccharomyces com- 90% algal removal of Aphanizomenon from a field reservoir prised the major microbial population in these EM mud balls, study by Schneider et al. (2015). Although the study by Park which, in conjunction with submerged , reduced total et al. has considerations and a variation of effectiveness in nitrogen and in water. Although mixed data have terms of cyanobacterial reduction percentages from different been collected in the laboratory and field studies in the report, studies, there is a demonstrated effectiveness of this method, there appear to be various factors influencing the success and with considerations and guidelines for further application in failure of the mentioned treatment interventions. This study larger-scale water . further questions the proposed effects of these treatments as For the purposes of this review, the focus is mainly on live they appear to be symptomatic treatments as opposed to ef- cell-to-cell interaction between the cyanobacteria and their fective controls of nuisance blooms (Lürling et al. 2016). predatory bacterial isolates, which, apart from the report of More effective results have been observed through the use of Lürling et al. (2016), is rather limited at a larger scale. metabolites from bacteria or the degradation of microcystins by specific bacteria (Yang et al. 2014), with possibilities for upscaling the treatments on a larger scale (Pei et al. 2007), in a Opinion on possible pitfalls controlled environment. A comprehensive review by Nybom (2013) lists various isolates that have been applied in the bacterial Areas that may require revision is the variation in culture degradation of microcystins and how these microcystins are methods and conditions between the predator and prey. The Appl Microbiol Biotechnol addition of a culture unaccustomed to the bloom conditions determining how effective a bacterium may be at effectively may affect the effectiveness of this culture and perhaps the reproducing the estimated inhibitory effects on cyanobacteria. required cell concentrations. This is indicated in the estimation The reasons listed above among the complex interactions of 6 to 7 days as the required time to present reasonable dam- within the bloom diversities may be among the reasons there age to cyanobacteria when bacterial isolates are concerned. is slow advancement in this area of research, with the remain- Moreover, the use of axenic strains in the biological control ing major question: why do these lytic agents fail to regulate studies may be cautioned if similar effects cannot be obtained bloom occurrences in nature? in the natural environment. The fact that these isolates almost never exist as axenic strains in a natural environment outside Compliance with ethical standards of the laboratory may indicate that the selected control agent needs to be effective within a mixed diversity. The changes Conflict of interest The authors declare that they have no conflict of that occur with gold standard isolates such as culture collec- interest. tions of Microcystis isolates may be giving an incorrect repre- Ethical statement This article does not contain any studies with sentation of the wild strains and possible outcomes with a performed by any of the authors. given control agent. This would require further research of control agent efficiency in different types of blooms, with other co-dominant species and the prospective impact on other References aquatic . Control agents need to be examined for their effects on Adam B, Klawonn I, Sveden JB, Bergkvist J, Nahar N, Walve J, Littmann a mixed population of cyanobacteria, which is the com- S, Whitehouse MJ, Lavik G, Kuypers MMM, Ploug H (2016) N2- mon finding in blooms, although certain species may be fixation, ammonium release and N-transfer to the microbial and classical food web within a community. 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